Studies on intact living tissues and cells often require the introduction of reagents or spectroscopic probes into the cells in order to provide a specific stimulus or to measure a particular cellular function. Such approaches are typically improved in both selectivity and sensitivity if the reagents or spectroscopic probes can be targeted to specific locations within a cell or organism. For example, the selective targeting of spectroscopic probes within a cell enables defined measurements of subcellular microenvironments such as within subcellular organelles to be precisely probed. The use of spectroscopic probes tagged to macromolecules enables spatio-temporal aspects of a tagged macromolecule to be monitored in real time in vivo. Such methods can be used to develop a variety of specific assays for cellular activation, or to create functional assays of enzymatic function. For example, the location of a nuclear receptor within a cell can be determined by creating a fusion protein of the receptor to a specific binding partner and then observing its movement, after addition of a fluorescent ligand for the specific binding partner, in response to a test stimulus, such as a chemical. In this example, activation of nuclear receptor results in their translocation into the nucleus, which can be used as an assay to determine the relative activity of a series of different chemicals.
In transgenic organisms the targeting of NMR contrast agents or positron emission probes enables whole organism imaging of specific tissues or cell types in the intact organism.
In spite of the many advantages of this approach there are few general methods of labeling macromolecules, such as proteins with organic compounds within intact living organisms, that are specific and selective enough to be of practical utility. Traditionally, labeling of proteins or polypeptides has been accomplished by chemical modification of purified proteins. For example, the normal procedures for fluorescent labeling required that the polypeptide be covalently reacted in vitro with a fluorescent dye then re-purified to remove excess dye and/or any damaged polypeptide. Using this approach, problems of labeling stoichiometry and disruption of biological activity are often encountered. Furthermore, the analysis of a chemically modified polypeptide within a cell, typically requires microinjection, or other means, to introduce the peptide into the cell. These methods are relatively inefficient, damaging to cells and not readily amenable to large numbers of cells, typically used for high throughput screening.
By contrast, the present invention provides a targeting method that combines the ability to genetically encoded a protein sequence, and specifically express that sequence within living cells, with the use of well characterized spectroscopic probes or other probes. The invention thus enables the use of a wide range of fluorophores, as well as other spectroscopic probes, including nuclear magnetic resonance (NMR), positron emission tomography (PET), relaxation reagents, chromophores, and other reagents such as chemical cross linkers, caged compounds, enzymatic substrates, activators and substrates to be selectively targeted within intact cells or organisms.
These advantages enable a range of whole cell based assays to be developed that provide specific advantages over, and are complementary with, existing methods of fluorescence analysis or in vivo labeling. For example, the utility of green fluorescent protein (GFP) based measurements are limited to fluorescence based measurements only. Furthermore, highly fluorescent GFP mutants are only available within a limited range of excitation and emission maxima, and typically have poorer fluorescent properties compared to the best small molecule fluorophores.
The use of FLASH, an arseno-fluorescein derivative, involves the addition of a membrane permeant arseno-fluorescein derivative to the cells that binds to a short alpha helix containing 4 cysteines that can be added to a protein of interest (Griffin, et al., (1998) Science 281 269–72). At present the FLASH approach is limited to a single fluorophore, however in combination with the present invention both methods may be used to specifically label two proteins with two different fluorophores.
In one embodiment, the invention provides for the intracellular expression of a high affinity specific binding partner that specifically interacts with a fluorophore-coupled ligand. In a preferred embodiment the specific binding partner comprises a single chain antibody (sFv) as the specific binding partner, and a hapten (phOx) as the ligand.